Project Summary Quorum sensing (QS) is a broadly distributed intercellular communication method used by bacteria to coordinate group activities. The opportunistic human pathogen Pseudomonas aeruginosa uses QS to regulate the production of numerous secreted products that include virulence factors, antibiotics, and proteases. The ability of P. aeruginosa to alter its behavior as a group contributes to the difficulty of treating this bacterium in diseases such as cystic fibrosis, in which it establishes chronic airway infections. In P. aeruginosa, QS is mediated in part by the transcription factors LasR and RhlR, which respond to acyl-homoserine lactone signals. In laboratory strains, LasR regulates RhlR, and together they control the transcription of hundreds of genes. LasR mutants are common in CF (although RhlR mutants are not usually observed) implying inactivation of QS in these isolates. However, in many P. aeruginosa isolates from cystic fibrosis, RhlR activity is not dependent on LasR and many LasR-null isolates still engage in acyl-homoserine lactone QS. The presence of active RhlR QS in these strains implies that QS is important for the regulation of certain factors within the CF lung. This proposal uses a large collection of P. aeruginosa isolates from cystic fibrosis patients to explore adaptations P. aeruginosa makes to preserve QS despite lasR mutation. The experiments described in this proposal ask: 1) what are the genes regulated by QS transcription factors and what is the ?core? QS regulon of clinical isolates; 2) what are the genetic changes that allow for LasR-independent RhlR activation in these clinical isolates and what are the direct gene targets of RhlR?; and 3) what are the molecular mechanisms that prevent RhlR mutants from emerging in populations of P. aeruginosa? The answers to these questions will give a more complete picture of QS in clinical P. aeruginosa, and guide efforts to target QS or QS-regulated genes to manage and treat bacterial populations in human disease.